The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

Measurement of NCX current with voltage-ramp protocol. As a standard pharmacological approach, a voltage-ramp protocol was used to measure the NCX current and to verify the differences in the two experimental groups containing 2 mM or 8 mM Na pip and to investigate the effectiveness of the selective NCX inhibitor ORM-10962. From a holding potential of − 40 mV, the membrane was depolarized to 30 mV with a slope of 0.7 V/s, then hyperpolarized to − 70 mV. The reverse mode was calculated at + 25 mV while the forward operation was calculated at − 60 mV, both during the downhill phase of the current. The pipette solution contained (in mM): 125 CsCl, 20 TEACl, 5 MgATP, 10 HEPES, 8 or 2 NaCl, titrated to pH 7.2 with CsOH. The free intracellular Ca 2+ was buffered to ~ 100 nM (by using an appropriate mixture of EGTA and Ca 2+ calculated by using MaxChelator software) to approximate a normal diastolic Ca 2+ value. The composition of the external solution was: 135 mM NaCl, 10 mM CsCl, 0.33 mM NaH 2 PO 4 , 10 mM TEACl, 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, 1 mM CaCl 2 , 20 µM ouabain, 50 µM lidocain, 1 µM nisoldipin, titrated to pH 7.4.

Measurement of Ni 2+ -sensitive current under predefined AP command. During the voltage
clamp experiments from Figs. 2, 3, 4 and 5, SN cells were paced using a previously recorded, canonical AP waveform. This AP waveform was obtained by average of 10 independent APs under perforated patch clamp conditions. The parameters of the AP command were: cycle length: 410 ms, maximal diastolic potential: − 57 mV, overshoot: 24 mV, action potential duration: 180 ms, diastolic depolarization slope: 0.124 mV/ms.
In the case of experiments demonstrated in Fig. 2, the NiCl 2 sensitive current under the predefined AP waveform was measured. The extracellular solution contained: 135 mM NaCl, 10 mM CsCl, 0.33 mM NaH 2 PO 4 , 10 mM TEACl, 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, 1.8 mM CaCl 2, 0.2 mM BaCl 2 , 20 µM ouabain, 50 µM lidocain, 1 µM nisoldipine, 1 µM mibefradil, titrated to pH 7.4. The intracellular solution contained (in mM): 125 CsCl, 20 TEACl, 5 MgATP, 10 HEPES and 10 EGTA titrated to pH 7.2 with CsOH, and 2 or 8 mM NaCl was added respectively. During these measurements, the intracellular Ca 2+ was unbuffered allowing Ca 2+ transients (CaT) that were monitored by Fluo-4 AM (5 µM) fluorescent dye. The isolated SN cells were loaded with the dye for 20 min at (a) Maximum diastolic potential (MDP) was calculated as the most negative potential reached before the AP depolarization. (b) Take off potential (TOP) defined as the voltage measured at the time when the voltage derivative exceeded 0.5 mV/ms. (c) The slope of depolarization was calculated as the mean voltage derivative of the AP between MDP and take off potential. (d) Action potential duration (APD) defined as the time interval between TOP and the next MDP. (e) Cycle length was measured between the peaks of two consecutive APs. (f) All experiments in this study were performed at 37 °C.

Measurements of Ca
Statistics. Normal distribution of the data was verified by using Shapiro-Wilk test. In this study, we used hierarchical analysis: the technical replicates obtained from the same heart were averaged providing n = 1. Therefore, the experimental number 'n' refers the number of hearts used. Thus, all experiments can be considered independent. For the experiments, 3-5 sinus node cells were used from each rabbit. Statistical significance (p < 0.05) was assessed using Student's t-test, or repeated measures ANOVA. Data are presented as mean ± S.E.M.

Modeling.
To mechanistically underpin our experimental findings, we conducted numerical simulations using the Maltsev  . Intracellular sodium and potassium concentrations are considered as constant in this model. The choice of model was motivated by a focus on fundamental mechanisms and the comparability to the experimental setting where potassium currents and I f were inhibited. We based our simulations on model #1, parameter set #4 as defined in 14 . The code was obtained from the CellML model repository and integrated using Matlab's ordinary differential equation solver ode15s (The Mathworks, Natick, MA, USA). All model codes are available in the Supplement.

Experimental validation of 2 and 8 mM [Na] pip groups.
In the first set of experiments conventional NCX-ramp protocol was used to study the absence and presence of a reverse current in 2 and 8 mM [Na] pip conditions, respectively. We also compared the effect of 1 µM ORM-10962 on the NCX current in the presence of 2 and 8 mM [Na] pip to exclude any Na-dependent action of ORM.
After registration of the control current (Fig. 1a, black traces), 1 µM ORM-10962 was applied (Fig. 1a, green traces) and finally 10 mM NiCl 2 (Fig. 1a, pink traces) was used to dissect the total NCX current. Panel b illustrates the ORM-10962 sensitive currents obtained from this experiment. In the case of 2 mM [Na] pip group (left side, blue curve) the current lacks the outward component. The total NCX current was calculated as a difference of the control and the Ni 2+ -insensitive current (Fig. 1c). As Fig. 1a  www.nature.com/scientificreports/ Indeed, the reversal potential in the 2 mM [Na] pip group is obviously far from the calculated value indicating that the intracellular Na + level sensed by the NCX was larger than the pipette Na + concentration. However, the NCX current was found to be negative from − 60 to + 30 mV in these experiments. Since our experiments were carried out within this range, we considered that reverse NCX is approximately absent in our experiments when 2 mM [Na] pip was employed. It is important to note that the inward component of the current (i.e., forward mode) in the presence of 8 mM [Na] pip did not differ from the current measured in the presence of 2 mM [Na] pip indicating that the operation of the forward modes was identical between groups.
Since we planned to use ORM to inhibit NCX (in the later part of the study), we considered important to exclude any possible Na + -dependent effect of the ORM prior to these experiments. The effect of ORM-10962 was calculated as a ratio between the total current (control vs 10 mM NiCl 2 ) and the ORM-inhibited fraction (control vs 1 µM ORM-10962). As Fig. 1d illustrates, we found no significant difference of the ORM effects between the 2 and 8 mM [Na] pip groups (2 mM fwd mode: 63.5 ± 8%, n = 6; 8 mM fwd mode: 74.5 ± 6%, n = 7; independent t-test). Since we did not observe reverse NCX in the presence of 2 mM NaCl, we could not quantify ORM effect in this case. The effect of ORM on the reverse mode in the presence of 8 mM [Na] pip was: 72.5 ± 5%, n = 7.
In the presence of 8 mM [Na] pip , both ORM-10962 and NiCl 2 dissected a notable outward current from the control, which may reflect the potential existence of reverse NCX that may be Na-dependent and increases as the membrane potential becomes more positive.
Therefore, based on these results, we consider the 8 mM [Na] pip group as an experimental condition where the reverse mode is active, and the 2 mM [Na] pip group as having suppressed or no reverse exchange activity. . The intracellular Ca 2+ was set to ~ 100 nM, K + and Ca 2+ currents were inhibited. In the presence of 2 mM [Na] pip (panel a, left side, repeated measures ANOVA) we did not find neither ORM-nor NiCl 2 -sensitive currents in the outward direction. In contrast, the outward component is clearly observable when 8 mM NaCl was used in the pipette (panel a, right side, repeated measures ANOVA). Panel (b) represents the ORM-10962 sensitive currents obtained from the experiment demonstrated in panel A. Current-voltage diagram (panel c) of the NiCl 2 -sensitive current demonstrates the lack of reverse current when 2 mM NaCl was employed in the internal solution (blue graph, independent t-test). Panel (d) illustrates comparison of the ORM-effect between experimental groups, and we found that 1 μM ORM-10962 similarly reduces the forward component of the NCX independent from the Na i level that may exclude pharmacological interactions between the applied Na i and ORM-10962 (independent t-test). Data shown as mean ± SEM, n = 7, * means p < 0.05. www.nature.com/scientificreports/ In the next set of experiments, we aimed to investigate whether the reverse NCX current develops under a SN action potential. In these experiments, a previously recorded canonical SN AP waveform was used (Fig. 2a) as command potential (see Methods for parameters), and the intracellular Ca 2+ was buffered by using 10 mM EGTA. The K + -currents, I CaL , I CaT , I Na/K were inhibited during the experiments. After recording the control current, the NCX current was estimated by application of 10 mM NiCl 2 (Fig. 2b) in order to fully inhibit the NCX. After current subtraction (Fig. 2c) in the presence of 2 mM [Na] pip , an outward current carrying 0.33 ± 0.3 pC was found (n = 5, capacitance: 51 ± 1 pF). In the presence of 8 mM [Na] pip , this net outward charge transport was significantly larger (2.1 ± 0.3 pC, n = 7, p < 0.05, capacitance: 52 ± 1 pF, independent t-test). This value is slightly smaller than the one predicted by the Maltsev-Lakatta model (2.45 pC).

Scientific
It is important to note that under this setting the Ca 2+ release was blocked due to Ca 2+ -channel inhibition and intracellular Ca 2+ buffering. It was necessary, since 10 mM NiCl 2 (concentration needed to complete inhibition of NCX) also suppresses the I CaL , which would seriously contaminate the results. Therefore, this method allows estimating the total carried charges through the reverse mode, however, lacks the Ca 2+ release, which is a crucial component of the NCX driving force. Thus, 1 µM ORM-10962 was used to explore the reverse NCX in the presence of Ca 2+ release ( Supplementary Fig. 1). Results indicate the existence of the reverse NCX as an outward ORM-sensitive current in the very beginning of the action potential, however, ORM-10962 only partially inhibit the NCX. Similarly, to the results with NiCl 2 , the outward component of the ORM-sensitive current was absent when 2 mM NaCl was applied in the patch pipette.

Without reverse NCX activity the Ca 2+ transient is smaller.
In the next set of experiments, we aimed to demonstrate the possible functional consequence of the active reverse NCX on the Ca 2+ transient magnitude.
In these experiments, we used again the canonical AP waveform (Fig. 3a) as command potential and nisoldipine and EGTA were omitted from the pipette solution. During control recordings we compared the diastolic Ca 2+  In the presence of active reverse mode the SR Ca 2+ content is increased. The larger Ca 2+ transient amplitude in the presence of active reverse NCX suggests increased SR Ca 2+ content. In order to address this question experimentally, we measured the SR Ca 2+ content by rapid application of 10 mM caffeine. Prior to caffeine administration, 10 consecutive AP commands were applied to reach a steady state Ca 2+ level of the SR. During caffeine flush, the membrane potential was kept constantly at − 80 mV. The SR Ca 2+ content was estimated by calculating the integral of the inward current in response to caffeine. As Fig. 4b  The activity of reverse NCX improves the "recovery" of Ca 2+ transients after caffeine application. It was hypothesized that the functional role of the reverse NCX could be further demonstrated during SR Ca 2+ refilling after a caffeine application. Thermodynamical considerations dictate, when SR is empty, the initially small Ca 2+ releases cause a negative shift of the NCX reversal potential. This may provide a large thermodynamical driving force for the reverse operation during the early phase of refilling. Therefore, an additional   Fig. 3b, the diastolic Ca 2+ did not show difference under steady state (14th: 3.2 ± 0.6 vs 2.2 ± 0.8; n = 13-13, independent t-test, Fig. 5b). In contrast, larger Ca 2+ transient amplitudes were found from the 2nd pulse when reverse mode operated (2nd: 1.25 ± 0.1 vs 1.00 ± 0.03, n = 13-15 respectively, p < 0.05, independent t-test; Fig. 5c).
Active reverse mode enhances pacemaker activity. In order to address the functional importance of reverse mode in spontaneous pacemaking we compared the AP and CaT characteristics between 2 and 8 mM [Na] pip groups obtained using the whole cell patch clamp configuration. The whole cell configuration was selected to provide identical experimental conditions as in previous experiments. As Fig. 6 demonstrates the illustrates original caffeine induced inward current, while panel (c) illustrates the Ca 2+ transients in the presence of 2 (blue trace) and 8 mM (red trace) pipette Na + . We found larger SR Ca 2+ content in presence of 8 mM pipette Na + (independent t-test, panel b). In contrast, we did not find statistically significant difference between the halfrelaxation times of the Ca 2+ transients (independent t-test, panel c). Data shown as mean ± SEM, n = 11 (panel b) and n = 17 (panel c), *means p < 0.05.  Fig. 6a-b). In line with this, the slope of diastolic depolarization was steeper when reverse mode was active (0.12 ± 0.02 mV/ms vs 0.07 ± 0.01 mV/ms; p < 0.05, n = 8-8, Fig. 6c). The APD was shorter in the 8 mM [Na] pip group (189 ± 3 ms vs 232 ± 11 ms; p < 0.05, n = 8-8, Fig. 6d In order to further validate AP data, perforated patch experiments were also performed with 2 and 8 mM NaCl in the pipette. The cycle length, APD and diastolic slope changed similarly between groups as was observed under experiments with the whole cell configuration ( Supplementary Fig. 2 and Table 1).

Facilitation of reverse mode further increases pacemaking.
In the second set of AP measurements, we aimed to facilitate reverse NCX function via Na/K pump inhibition mediated Na + i increase by using the perforated patch configuration. To inhibit Na/K pump, 1 µM strophantin was employed.
The Maltsev-Lakatta "minimal model" confirms an important role for Ca 2+ influx through reverse NCX. Maltsev   www.nature.com/scientificreports/ This minimal model was used to assess the effects of Na i changes on AP cycle length, intracellular Ca 2+ levels, as well as NCX and I CaL (Fig. 8). We found that in this minimal model with a reduced set of ionic currents and fixed Na i and K i , at least 6 mM Na + i is required for sustained pacemaking and that an increase of Na + i from 6 to 10 mM decreases the AP cycle length (1011 ms → 450 ms, Fig. 8a first row) after transient changes had equilibrated. In line with this, a gradual increase of reverse NCX occurred (Fig. 8a second row, positive current), together with an increase of the Ca 2+ transient amplitude and diastolic Ca 2+ levels ( Fig. 8a third row) as well as network SR Ca 2+ levels ( Fig. 8a fourth row) that are in agreement with our experimental observations.
It is important to note that the peak I CaL magnitude gradually decreased as Na + i increased (6 mM Na + i : − 16.5 pA/pF; 8 mM Na + i : − 15.1 pA/pF; 10 mM Na + i : − 12.6 pA/pF), however the carried charge, in contrast, increased   Fig. 8a last row). Table 2 summarizes the contributions of forward and reverse NCX as well as I CaL to intracellular calcium cycling. The time spent in forward mode is 2.3-5.1 × longer than that spent in reverse mode and the charge carried is 2.5-3.1 × bigger. I CaL carries 2.9-4.2 × more charge than reverse NCX but only 1.5-2.1 × more ions. The relative contribution of reverse NCX is bigger under high Na + i conditions. The role of reverse NCX in SN pacemaking was further elucidated by disabling reverse NCX in the model (setting NCX to zero whenever it was positive, brown traces in Fig. 8) and thus consider only forward NCX. A marked loss of SR Ca 2+ content was observed (Fig. 8). Pacemaking could not be sustained without reverse NCX in the model. While pacemaking was observed for some beats for higher Na + i levels, it ceased eventually (after < 10 s for all investigated Na + i levels), suggesting that Ca 2+ influx through reverse mode of the exchanger is essential for maintaining a stable SR Ca 2+ level, or in other words, the Ca 2+ influx provided by the I CaL per se is insufficient to maintain pacemaking in this model.

Discussion
In this study we characterized the reverse NCX and its functional role during the SN action potential. In accordance with previous numerical simulations 14 (1) a voltage-, and Na + i dependent outward current was found during the initial part of the SN action potential which was sensitive to ORM-10962 and NiCl 2 . (2) Higher SR Ca 2+ content and (3) faster heart rate was observed in the presence of active reverse NCX.

Does the reverse mode of NCX exist in SN cells?
The Na + /Ca 2+ exchanger of the cardiac sarcolemma transporting 3 Na + for 1 Ca 2+ represents the main Ca 2+ extrusion mechanism of the cell. The NCX exerts a ther-  www.nature.com/scientificreports/ modynamically defined reversal potential (E NCX ) based on the transport stoichiometry: E NCX = 3E Na − 2E Ca . This means when the actual membrane potential (i.e.: action potential) is more positive than E NCX , Ca 2+ entry and consequential outward current happens via the reverse mode of NCX. When membrane potential falls below E NCX , the direction of the transport changes to Ca 2+ efflux and inward current is carried by forward mode. Therefore, the presence or absence of reverse Na + /Ca 2+ exchange in a given cell type is defined by the actual driving force of the NCX. Based on previous model simulations 14 , reverse operation of the NCX is expected when the membrane potential is more positive than 0 mV. When our experimental conditions were used for calculations to approximate the NCX equilibrium, the reverse mode was favoured in the first 55-65 ms from the AP upstroke ( Supplementary Fig. 1a). In line with this calculation, application of NiCl 2 dissected an outward current in this range when the reverse mode was active (i.e.: Na + i was set to 8 mM in the pipette) but in the presence of 2 mM Na + i the outward current was suppressed (Fig. 2). This result indicates that the reverse mode of the NCX could be able to develop under a SN AP, therefore the SN membrane potential theoretically enables operation of the reverse NCX.
In order to examine the development of reverse mode during working Ca 2+ -handling we also determined the NCX current as an ORM-sensitive current under canonical AP waveform as command potential (Supplementary Fig. 1b-c). Since the ORM-sensitive current is very susceptible to any spontaneous current change, we measured ORM-effects in separated groups to avoid any shortcomings from time dependent spontaneous decline of I Ca . The ORM sensitive current also revealed an outward current component at positive membrane potentials, when Na + i was 8 mM. Taking together, these results support previous modelling simulations 14 suggesting an existing reverse NCX in SN cells by the following experimental reasons: (1) the Ni 2+ -and ORM-sensitive outward current component was only detectable in the presence of 8 mM pipette Na + and disappeared when low Na + was applied. This indicates that the outward component of the Ni 2+ -or ORM-sensitive current is Na + i dependent. (2) As it was thermodynamically predicted, this outward component of the Ni 2+ -or ORM-sensitive current appeared only at positive membrane potentials, during the very first section of the AP.
How these findings in rabbit SN cells and models relate to human SN cells 21 and models 22,23 remains to be studied.

Does reverse NCX activity modulate SN pacemaking?
Considering that SN pacemaking is critically based on the actual SR Ca 2+ content, it may imply an important functional role of reverse NCX in cardiac SN pacemaking mechanism.
In line with these numerical calculations, in our experiments a larger CaT amplitude was found (seen in Fig. 3b) as a consequence of larger SR Ca 2+ content in the presence of active reverse NCX (Fig. 4). Since the larger SR Ca 2+ content generates larger Ca 2+ release, NCX takes more time to extrude the Ca 2+ providing longer decay in the case of 8 mM Na + i group. However, this value did not reach statistical significance possibly due to the larger variance of the data. These changes could be attributable to the "extra" Ca 2+ influx from the reverse exchange activity. The functional importance of reverse NCX was further explored during "recovery" of Ca 2+ homeostasis after caffeine application (Fig. 5). The caffeine flush empties the SR, therefore reapplication of the stimuli, the Ca 2+ i will be lower during the first steps (i.e.: "Ca 2+ load"). Based on thermodynamical considerations, the lower Ca 2+ i levels during the couple of first stimuli shifts the NCX equilibrium toward more negative values favouring an initially large, than gradually decreasing outward NCX parallel with the increase of Ca 2+ i . It is supposed that this initially large reverse component may boost up the recovery of CaT after caffeine application. As Fig. 5 illustrates, a step-by-step increasing diastolic Ca 2+ level and CaT amplitude was found. Taken together these results, an important functional role of reverse NCX could be considered since: (1) the Ca 2+ transient amplitude was higher in the presence of active reverse mode and (2) in line with this, the SR Ca 2+ content increased. (3) The cells with active reverse exchange exerted faster refilling of Ca 2+ in the initial steps after caffeine application, indicating synergistic effect between reverse NCX and I Ca during SR Ca 2+ loading. Table 2. Model results for varying levels of intracellular sodium. Cycle length of spontaneous action potential (AP) initiation, time NCX spends in forward (tForward) and reverse (tReverse) mode during each AP, charge carried by NCX in forward (QForward) and reverse (QReverse) mode, carried by I CaL (QICaL) and ratios between them including the ratio of ions carried by I CaL (nICaL) and NCX in reverse mode (nReverse).  Figs. 1, 2, 3, 4 and 5 indicated an existing reverse NCX that could provide additional Ca 2+ influx to the cell in each cycle. Since SN pacemaking is largely based on Ca 2+ i , a potential role of reverse NCX in setting the actual SN AP cycle length was expected. When spontaneous APs were measured in the presence of 2 and 8 mM Na pip we found shorter cycle length and steeper slope when reverse NCX was active. The parallel measured higher Ca 2+ transient amplitude in this group indicates again that additional Ca 2+ influx through active reverse mode facilitates pacemaking rate via shifting the Ca 2+ i level (Fig. 6). Interestingly, the APD was also shortened in the presence of 8 mM Na pip. This could be a simple consequence of the higher Ca 2+ i -induced faster I CaL inactivation, I NaK , and/or due to an additional repolarizing current via possible activation of the small-conductance Ca 2+ -activated K + -channel, as was demonstrated elegantly in a previous study 24 .
Computer simulations using the Maltsev-Lakatta minimal model suggest that spontaneous pacemaking fails at low Na i levels (2 mM) and in the absence of reverse NCX (Fig. 8a,b panels). In contrast, spontaneous pacemaking was observed even in the 2 mM Na pip group, i.e. with the inactive reverse NCX in our in vitro experiments. This discrepancy could be due to the fact that in the model the I CaL current density gradually decreases as Na i increases. Similarly, I CaL integral is smaller when reverse NCX is absent in the model. Since in our experiments no Na + i dependent I CaL behaviour was observed ( Supplementary Fig. 3), we suggest that Ca 2+ influx through I CaL did not change significantly and still provided sufficient loading for the SR in the experiments when reverse mode was suppressed by 2 mM Na pip .
Previous experiments with digoxigenin indicated that moderate increase in Na + i shortens the cycle length parallel with increase of Ca 2+ and arrhythmic behavior 25,26 . These results and our observations suggest that amplification of reverse NCX could further increase the spontaneous pacemaking. In our experiments, 1 µM strophantin was employed to increase Na + i via Na/K-ATPase block. The increasing Na + i is expected to shift E NCX towards more negative values such that the reverse component of the NCX increases. In line with this, a higher pacemaking rate was found in response to strophantin administration (Fig. 7a). In contrast, the APD remained unchanged after strophantin. However, the Na/K pump generates net outward current, it is feasible that the increased Ca 2+ i shortens the APD and these opposite effects lead to negligible change in the APD. To further confirm the role of reverse NCX in this process, we inhibited the exchanger prior to strophantin administration. In the presence of NCX inhibition strophantin failed to alter the spontaneous automaticity, which could be the consequence of the unchanged intracellular Ca 2+ i level after strophantin application in the presence of reverse NCX block (Fig. 7b). This result is in agreement with previous study on canine ventricular myocytes where strophantin-mediated spontaneous diastolic Ca 2+ releases could be suppressed when the cells were previously treated with 1 µM ORM-10103 27 .
One can speculate that the observed results could be attributable to the NCX forward mode activity. Theoretically, the reduced Na i (i.e. 2 mM Na pip ) facilitates forward NCX leading to net Ca 2+ loss while higher Na + -level inhibits the forward mode causing net Ca 2+ gain. Therefore, in the case of 8 mM Na pip both the Na + -mediated reverse mode activation and forward mode suppression could be able to increase the Ca 2+ i . However, in SN the forward mode activity has considerable contribution to the diastolic depolarization. Therefore, if Ca 2+ increase was caused by forward mode inhibition the diastolic slope would decrease as an indicator of inward current reduction.
Numerical simulations indicated progressively increasing heart rate as Na + i increases. As Fig. 8 illustrates, both reverse NCX and the charge carried by I CaL are increased and could account for the accelerated pacemaking. However, as Fig. 8 demonstrates, the suppression of the reverse component causes a great loss in the SR Ca 2+ content and abrupt termination of spontaneous pacemaking. Obviously, the charge carried by I CaL also reduced in this case, but it may imply that Ca 2+ influx by the reverse activity contributes in setting the SR Ca 2+ content, suggesting important indirect role in setting the actual heart rate. This is underpinned by the fact that SR Ca 2+ loss and failure of automaticity could be rescued by improved SERCA activity ( Supplementary Fig. 4). This result may further support that Ca 2+ influx through reverse exchange provides a functionally important fraction of the total SR Ca 2+ content, i.e. the reverse NCX may indirectly contribute to fine tuning of the heart rate.

Is the reverse NCX more important in SN than in ventricular cells?
The exact role of the reverse NCX in ventricular myocytes is not fully clarified under normal circumstances. Initial studies suggested that reverse mode could trigger Ca 2+ release 28,29 however this was later questioned 30 . Further studies claimed that reverse Ca 2+ influx is able to augment the Ca 2+ transient via a synergistic interaction with the I CaL that may depend on the actual Na + i level [31][32][33][34][35] . Based on these experimental results, the reverse mode in ventricular myocytes is expected to facilitate the Ca 2+induced Ca 2+ release by improving its efficacy however it seems no essential for the normal Ca 2+ handling.
The Ca 2+ influx of the reverse NCX and I CaL can be calculated and compared between ventricular and SN myocytes using respective computational models. For the Mahajan rabbit ventricular myocyte model 36 (dynamic Na + i ranging between 11.4 and 11.5 mM in silico; in vitro range: 9-11.5 mM 37 ), the I CaL /reverse NCX Ca 2+ influx ratio is 17.2 compared to 3.3 found in this study for SN cells (at Na + i of 8 mM) under in silico condition, and 2.81 under in vitro condition.
This large discrepancy between ventricular myocytes and SN cells could be the consequence of the long plateau phase in the ventricular cells providing long-lasting opening of the I CaL and a maintained Ca 2+ influx. In SN cells, the absence of the plateau phase considerably restricts the I CaL opening time limiting I CaL Ca 2+ influx. This may explain that under normal condition, in the ventricular myocytes, the I CaL is markedly dominating over the reverse NCX but it may represent an important source of Ca 2+ influx in SN cells since the Ca 2+ entry via I CaL is restricted due to the characteristic SN AP waveform. www.nature.com/scientificreports/ Electrical heterogeneity within the SN. There is a large body of evidence that the SN exerts considerable electrical heterogeneity (reviewed by: 38 ). The action potentials recorded from the central region markedly differ from the action potentials obtained from the transitional or peripheral zone 39 . In context of the reverse mode, two important differences should be mentioned: from the centre region to the distal areas the (1) action potential upstroke becomes significantly larger and (2) the cycle length shortens. These differences could be explained by the influence of the atrial muscle 40 , the higher role of I f in the periphery 41,42 , and the different role of the I CaL in pacemaking from the centre to the periphery 39,43 .
Considering the results of this study, it is feasible that the small upstroke in the centre region considerably restricts the reverse function, while the distal areas may enable increasing amount of reverse mode. This increasing reverse NCX gradient from the centre to the periphery may contribute in the observed AP cycle length shortening in the transitional zone and the periphery, therefore the reverse NCX could contribute to the electrophysiological heterogeneity of the SN.

Conclusion
In this study, we identified a Na + -and voltage-dependent, ORM-10962 and Ni 2+ -sensitive outward current component appearing during the early SN action potential that could be considered as reverse NCX.
Our results suggest that Ca 2+ influx through the reverse NCX may contribute to the setting of the SR Ca 2+ content and might therefore represent an additional mechanism of the SN coupled-clock pacemaking system that contributes to the control of the heart rate in SN cells.

Study limitations
(1) It is important to note that ORM-sensitive currents (in Supplementary Fig. 1) do not allow to calculate current parameters (amplitude, time, carried charges) due to 2 reasons: (a) 1 µM ORM-10962 only partially inhibits the NCX. (b) ORM-sensitive current is very susceptible to any spontaneous current change, thus we measured ORM-effects in separated groups to avoid any shortcomings from time dependent spontaneous decline of I Ca . Considering these limitations, the aim of the ORM-sensitive current was to demonstrate the existence of the reverse NCX current during the initial phase of the AP. (2) In the experiments, we used only one canonical action potential waveform. Since the SN is a heterogeneous system, our results can be interpreted as representative for SN cells that have a relatively large action potential overshoot and should not be generalized to the entire SN. (3) A possible contribution of the I Na may change the intracellular Na + -level of the cells that could influence the function of the NCX. (4) In the experiments, we used 2 and 8 mM NaCl in the patch pipette. This change in the intracellular Na + level could also influence the I Na , Na/K pump, Na/H pump and I f .

Data availability
All data generated or analysed during this study are included in this published article [and its supplementary information files].